Human extracellular signal regulated kinases

ABSTRACT

ERK-7 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing ERK-7 polypeptides and polynucleotides in diagnostic assays.

FIELD OF THE INVENTION

[0001] This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides sometimes hereinafterreferred to as novel human extracellular signal regulated kinases(ERK-7), to their use in diagnosis and in identifying compounds that maybe agonists, antagonists that are potentially useful in therapy, and toproduction of such polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

[0002] The drug discovery process is currently undergoing a fundamentalrevolution as it embraces “functional genomics”, that is, highthroughput genome- or gene-based biology. This approach as a means toidentify genes and gene products as therapeutic targets is rapidlysuperseding earlier approaches based on “positional cloning”. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

[0003] Functional genomics relies heavily on high-throughput DNAsequencing technologies and the various tools of bioinformatics toidentify gene sequences of potential interest from the many molecularbiology databases now available. There is a continuing need to identifyand characterise further genes and their related polypeptides/proteins,as targets for drug discovery.

SUMMARY OF THE INVENTION

[0004] The present invention relates to ERK-7, in particular ERK-7polypeptides and ERK-7 polynucleotides, recombinant materials andmethods for their production. Such polypeptides and polynucleotides areof interest in relation to methods of treatment of certain diseases,including, but not limited to, cancers, especially of the breast, ovary,colon, kidney and pancreas and inflammatory diseases hereinafterreferred to as “diseases of the invention”. In a further aspect, theinvention relates to methods for identifying agonists and antagonists(e.g., inhibitors) using the materials provided by the invention, andtreating conditions associated with ERK-7 imbalance with the identifiedcompounds. In a still further aspect, the invention relates todiagnostic assays for detecting diseases associated with inappropriateERK-7 activity or levels.

DESCRIPTION OF THE INVENTION

[0005] In a first aspect, the present invention relates to ERK-7polypeptides. Such polypeptides include:

[0006] (a) a polypeptide encoded by a polynucleotide comprising thesequence of SEQ ID NO:1;

[0007] (b) a polypeptide comprising a polypeptide sequence having atleast 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence ofSEQ ID NO:2;

[0008] (c) a polypeptide comprising the polypeptide sequence of SEQ IDNO:2;

[0009] (d) a polypeptide having at least 95%, 96%, 97%, 98%, or 99%identity to the polypeptide sequence of SEQ ID NO:2;

[0010] (e) the polypeptide sequence of SEQ ID NO:2; and

[0011] (f) A polypeptide having or comprising a polypeptide sequencethat has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 comparedto the polypeptide sequence of SEQ ID NO:2;

[0012] (g) fragments and variants of such polypeptides in (a) to (f).

[0013] Polypeptides of the present invention are believed to be membersof the extracellular signal regulated kinase family of polypeptides.They are therefore of interest because This invention describes theseventh member of the ERK family in humans, revealed in a RDA analysisof larynx tumor tissue. It is homologous to the seventh ERK familymember of rat at both the nucleotide and translated protein level.Electronic Northern analysis of cDNA fragments from the novel ERK-7 showthat it is almost exclusively expressed in transformed tissue. Theelectronic Northern finds Erk-7 prinarily in breast, ovarian and larynxcarcinomas.

[0014] Six human extracellular signal regulated kinases (ERKs) have beenidentified. They are involved in the transmission of the extracellularsignals by which growth factor receptors to activate genetranscription—thus, ERK-1 lies in the Ras-MAP Kinase pathway andactivates by phosphorylation transcription factors such as Jun and Fos.

[0015] Rat Erk-7, in contrast to Erk-1-6, is not regulated by commonextracellular signals, but by the interactions of the C-terminal proteindomain with intracellular machinery. The amino terminal of the humanhomologue is as yet incomplete—but the existing sequence contains a welldefined C-terminal domain and some 70 amino acids homologous to theC-terminal of the rat Erk-7 kinase domain. The C-terminal domain isthought to be responsible for localization, activation and cell growth(Abe et al., 1999),

[0016] The established biological role of Erks, the homology of thediscovery to rat Erk7, and its restricted distribution in canceroustissues suggests that Erk-7 is a protein with an important role incancer development, and a potential therapeutic target.

[0017] The biological properties of the ERK-7 are hereinafter referredto as “biological activity of ERK-7” or “ERK-7 activity”. Preferably, apolypeptide of the present invention exhibits at least one biologicalactivity of ERK-7.

[0018] Polypeptides of the present invention also includes variants ofthe aforementioned polypeptides, including all allelic forms and splicevariants. Such polypeptides vary from the reference polypeptide byinsertions, deletions, and substitutions that may be conservative ornon-conservative, or any combination thereof, Particularly preferredvariants are those in which several, for instance from 50 to 30, from 30to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to1 or 1 amino acids are inserted, substituted, or deleted, in anycombination.

[0019] Preferred fragments of polypeptides of the present inventioninclude an isolated polypeptide comprising an amino acid sequence havingat least 30, 50 or 100 contiguous amino acids from the amino acidsequence of SEQ ID NO: 2, or an isolated polypeptide comprising an aminoacid sequence having at least 30, 50 or 100 contiguous amino acidstruncated or deleted from the amino acid sequence of SEQ ID NO: 2.Preferred fragments are biologically active fragments that mediate thebiological activity of ERK-7, including those with a similar activity oran improved activity, or with a decreased undesirable activity. Alsopreferred are those fragments that are antigenic or immunogenic in ananimal, especially in a human.

[0020] Fragments of the polypeptides of the invention may be employedfor producing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention. Thepolypeptides of the present invention may be in the form of the “mature”protein or may be a part of a larger protein such as a precursor or afusion protein. It is often advantageous to include an additional aminoacid sequence that contains secretory or leader sequences,pro-sequences, sequences that aid in purification, for instance multiplehistidine residues, or an additional sequence for stability duringrecombinant production.

[0021] Polypeptides of the present invention can be prepared in anysuitable manner, for instance by isolation form naturally occurringsources, from genetically engineered host cells comprising expressionsystems (vide infra) or by chemical synthesis, using for instanceautomated peptide synthesizers, or a combination of such methods. Meansfor preparing such polypeptides are well understood in the art.

[0022] In a further aspect, the present invention relates to ERK-7polynucleotides. Such polynucleotides include:

[0023] (a) a polynucleotide comprising a polynucleotide sequence havingat least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotidesequence of SEQ ID NO:1;

[0024] (b) a polynucleotide comprising the polynucleotide of SEQ IDNO:1;

[0025] (c) a polynucleotide having at least 95%, 96%, 97%, 98%, or 99%identity to the polynucleotide of SEQ ID NO:1;

[0026] (d) the isolated polynucleotide of SEQ ID NO:1;

[0027] (e) a polynucleotide comprising a polynucleotide sequenceencoding a polypeptide sequence having at least 95%, 96%, 97%, 98%, or99% identity to the polypeptide sequence of SEQ ID NO:2;

[0028] (f) a polynucleotide comprising a polynucleotide sequenceencoding the polypeptide of SEQ ID NO:2;

[0029] (g) a polynucleotide having a polynucleotide sequence encoding apolypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identityto the polypeptide sequence of SEQ ID NO:2;

[0030] (h) a polynucleotide encoding the polypeptide of SEQ ID NO:2;

[0031] (i) a polynucleotide having or comprising a polynucleotidesequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99compared to the polynucleotide sequence of SEQ ID NO:1;

[0032] (j) a polynucleotide having or comprising a polynucleotidesequence encoding a polypeptide sequence that has an Identity Index of0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence ofSEQ ID NO:2; and

[0033] polynucleotides that are fragments and variants of the abovementioned polynucleotides or that are complementary to above mentionedpolynucleotides, over the entire length thereof.

[0034] Preferred fragments of polynucleotides of the present inventioninclude an isolated polynucleotide comprising an nucleotide sequencehaving at least 15, 30, 50 or 100 contiguous nucleotides from thesequence of SEQ ID NO: 1, or a polynucleotide comprising an sequencehaving at least 30, 50 or 100 contiguous nucleotides truncated ordeleted from the sequence of SEQ ID NO: 1.

[0035] Preferred variants of polynucleotides of the present inventioninclude splice variants, allelic variants, and polymorphisms, includingpolynucleotides having one or more single nucleotide polymorphisms(SNPs).

[0036] Polynucleotides of the present invention also includepolynucleotides encoding polypeptide variants that comprise the aminoacid sequence of SEQ ID NO:2 and in which several, for instance from 50to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3to 2, from 2 to 1 or 1 amino acid residues are substituted, deleted oradded, in any combination.

[0037] In a further aspect, the present invention providespolynucleotides that are RNA transcripts of the DNA sequences of thepresent invention. Accordingly, there is provided an RNA polynucleotidethat:

[0038] (a) comprises an RNA transcript of the DNA sequence encoding thepolypeptide of SEQ ID NO:2;

[0039] (b) is the RNA transcript of the DNA sequence encoding thepolypeptide of SEQ ID NO:2;

[0040] (c) comprises an RNA transcript of the DNA sequence of SEQ IDNO:1; or

[0041] (d) is the RNA transcript of the DNA sequence of SEQ ID NO:1;

[0042] and RNA polynucleotides that are complementary thereto.

[0043] The polynucleotide sequence of SEQ ID NO:1 shows homology withAbe, M. K., Kuo, W. L., Hershenson, M. B., and Rosner, M. R. (1999).Extracellular signal-regulated kinase 7 (ERK7), a novel ERK with aC-terminal domain that regulates its activity, its cellularlocalization, and cell growth. MOLECULAR.AND.CELLULAR.BIOLOGY. 19,1301-1312.

[0044] The polynucleotide sequence of SEQ ID NO:1 is a cDNA sequencethat encodes the polypeptide of SEQ ID NO:2. The polynucleotide sequenceencoding the polypeptide of SEQ ID NO:2 may be identical to thepolypeptide encoding sequence of SEQ ID NO:1 or it may be a sequenceother than SEQ ID NO:1, which, as a result of the redundancy(degeneracy) of the genetic code, also encodes the polypeptide of SEQ IDNO:2. The polypeptide of the SEQ ID NO:2 is related to other proteins ofthe Erk family, having homology and/or structural similarity with RatErk-7 (Abe, M. K., Kuo. W. L., Hershenson, M. B., and Rosner, M. R.(1999). Extracellular signal-regulated kinase 7 (ERK7), a novel ERK witha C-terminal domain that regulates its activity, its cellularlocalization, and cell growth. MOLECULAR.AND.CELLULAR.BIOLOGY. 19,1301-1312).

[0045] Preferred polypeptides and polynucleotides of the presentinvention are expected to have, inter alia, similar biologicalfunctions/properties to their homologous polypeptides andpolynucleotides. Furthermore, preferred polypeptides and polynucleotidesof the present invention have at least one ERK-7 activity.

[0046] Polynucleotides of the present invention may be obtained usingstandard cloning and screening techniques from a cDNA library derivedfrom mRNA in cells of human primary breast cancer, colon adenocarcinoma,peripheral blood neutrophils and ovarian adenocarcinoma, (see forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)). Polynucleotides of the invention can also be obtained fromnatural sources such as genomic DNA libraries or can be synthesizedusing well known and commercially available techniques.

[0047] When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself, or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence that facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

[0048] Polynucleotides that are identical, or have sufficient identityto a polynucleotide sequence of SEQ ID NO:1, may be used ashybridization probes for cDNA and genomic DNA or as primers for anucleic acid amplification reaction (for instance, PCR). Such probes andprimers may be used to isolate full-length cDNAs and genomic clonesencoding polypeptides of the present invention and to isolate cDNA andgenomic clones of other genes (including genes encoding paralogs fromhuman sources and orthologs and paralogs from species other than human)that have a high sequence similarity to SEQ ID NO:1, typically at least95% identity. Preferred probes and primers will generally comprise atleast 15 nucleotides, preferably, at least 30 nucleotides and may haveat least 50, if not at least 100 nucleotides. Particularly preferredprobes will have between 30 and 50 nucleotides. Particularly preferredprimers will have between 20 and 25 nucleotides.

[0049] A polynucleotide encoding a polypeptide of the present invention,including homologs from species other than human, may be obtained by aprocess comprising the steps of screening a library under stringenthybridization conditions with a labeled probe having the sequence of SEQID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides;and isolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan. Preferred stringent hybridization conditionsinclude overnight incubation at 420° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0×SSC at about 650° C. Thus the present invention alsoincludes isolated polynucleotides, preferably with a nucleotide sequenceof at least 100, obtained by screening a library under stringenthybridization conditions with a labeled probe having the sequence of SEQID NO:1 or a fragment thereof, preferably of at least 15 nucleotides.

[0050] The skilled artisan will appreciate that, in many cases, anisolated cDNA sequence will be incomplete, in that the region coding forthe polypeptide does not extend all the way through to the 5′ terminus.This is a consequence of reverse transcriptase, an enzyme withinherently low “processivity” (a measure of the ability of the enzyme toremain attached to the template during the polymerisation reaction),failing to complete a DNA copy of the mRNA template during first strandcDNA synthesis.

[0051] There are several methods available and well known to thoseskilled in the art to obtain full-length cDNAs, or extend short cDNAs,for example those based on the method of Rapid Amplification of cDNAends (RACE) (see, for example, Frohman et al., Proc Nat Acad Sci USA 85,8998-9002, 1988). Recent modifications of the technique, exemplified bythe Marathon (trade mark) technology (Clontech Laboratories Inc.) forexample, have significantly simplified the search for longer cDNAs. Inthe Marathon (trade mark) technology, cDNAs have been prepared from mRNAextracted from a chosen tissue and an ‘adaptor’ sequence ligated ontoeach end. Nucleic acid amplification (PCR) is then carried out toamplify the “missing” 5′ end of the cDNA using a combination of genespecific and adaptor specific oligonucleotide primers. The PCR reactionis then repeated using ‘nested’ primers, that is, primers designed toanneal within the amplified product (typically an adapter specificprimer that anneals further 3′ in the adaptor sequence and a genespecific primer that anneals further 5′ in the known gene sequence). Theproducts of this reaction can then be analyzed by DNA sequencing and afull-length cDNA constructed either by joining the product directly tothe existing cDNA to give a complete sequence, or carrying out aseparate full-length PCR using the new sequence information for thedesign of the 5′ primer.

[0052] Recombinant polypeptides of the present invention may be preparedby processes well known in the art from genetically engineered hostcells comprising expression systems. Accordingly, in a further aspect,the present invention relates to expression systems comprising apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

[0053] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof forpolynucleotides of the present invention. Polynucleotides may beintroduced into host cells by methods described in many standardlaboratory manuals, such as Davis et al., Basic Methods in MolecularBiology (1986) and Sambrook et al.(ibid). Preferred methods ofintroducing polynucleotides into host cells include, for instance,calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, micro-injection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introduction orinfection.

[0054] Representative examples of appropriate hosts include bacterialcells, such as Streptococci, Staphylococci, E. coli, Streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 andBowes melanoma cells; and plant cells.

[0055] A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes from insertion elements, from yeast chromosomal elements,from viruses such as baculoviruses, papova viruses, such as SV40,vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies virusesand retroviruses, and vectors derived from combinations thereof, such asthose derived from plasmid and bacteriophage genetic elements, such ascosmids and phagemids. The expression systems may contain controlregions that regulate as well as engender expression. Generally, anysystem or vector that is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate polynucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., (ibid). Appropriatesecretion signals may be incorporated into the desired polypeptide toallow secretion of the translated protein into the lumen of theendoplasmic reticulum, the periplasmic space or the extracellularenvironment. These signals may be endogenous to the polypeptide or theymay be heterologous signals.

[0056] If a polypeptide of the present invention is to be expressed foruse in screening assays, it is generally preferred that the polypeptidebe produced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

[0057] Polypeptides of the present invention can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high performance liquid chromatography is employed forpurification. Well known techniques for refolding proteins may beemployed to regenerate active conformation when the polypeptide isdenatured during intracellular synthesis, isolation and/or purification.

[0058] Polynucleotides of the present invention may be used asdiagnostic reagents, through detecting mutations in the associated gene.Detection of a mutated form of the gene characterized by thepolynucleotide of SEQ ID NO:1 in the cDNA or genomic sequence and whichis associated with a dysfunction will provide a diagnostic tool that canadd to, or define, a diagnosis of a disease, or susceptibility to adisease, which results from under-expression, over-expression or alteredspatial or temporal expression of the gene. Individuals carryingmutations in the gene may be detected at the DNA level by a variety oftechniques well known in the art.

[0059] Nucleic acids for diagnosis may be obtained from a subject'scells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genomic DNA may be used directly for detection or it maybe amplified enzymatically by using PCR, preferably RT-PCR, or otheramplification techniques prior to analysis. RNA or cDNA may also be usedin similar fashion. Deletions and insertions can be detected by a changein size of the amplified product in comparison to the normal genotype.Point mutations can be identified by hybridizing amplified DNA tolabeled ERK-7 nucleotide sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase digestion or bydifferences in melting temperatures. DNA sequence difference may also bedetected by alterations in the electrophoretic mobility of DNA fragmentsin gels, with or without denaturing agents, or by direct DNA sequencing(see, for instance, Myers et al., Science (1985) 230:1242). Sequencechanges at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (see Cotton et al., Proc Natl Acad Sci USA (1985) 85:4397-4401).

[0060] An array of oligonucleotides probes comprising ERK-7polynucleotide sequence or fragments thereof can be constructed toconduct efficient screening of e.g., genetic mutations. Such arrays arepreferably high density arrays or grids. Array technology methods arewell known and have general applicability and can be used to address avariety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability, see, for example, M. Chee etal., Science, 274, 610-613 (1996) and other references cited therein.

[0061] Detection of abnormally decreased or increased levels ofpolypeptide or mRNA expression may also be used for diagnosing ordetermining susceptibility of a subject to a disease of the invention.Decreased or increased expression can be measured at the RNA level usingany of the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, nucleic acid amplification, forinstance PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods. Assay techniques that can be used to determinelevels of a protein, such as a polypeptide of the present invention, ina sample derived from a host are well-known to those of skill in theart. Such assay methods include radio-immunoassays, competitive-bindingassays, Western Blot analysis and ELISA assays.

[0062] Thus in another aspect, the present invention relates to adiagnostic kit comprising:

[0063] (a) a polynucleotide of the present invention, preferably thenucleotide sequence of SEQ ID NO: 1, or a fragment or an RNA transcriptthereof;

[0064] (b) a nucleotide sequence complementary to that of (a);

[0065] (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO:2 or a fragment thereof; or

[0066] (d) an antibody to a polypeptide of the present invention,preferably to the polypeptide of SEQ ID NO:2.

[0067] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, particularlydiseases of the invention, amongst others.

[0068] The polynucleotide sequences of the present invention arevaluable for chromosome localisation studies. The sequence isspecifically targeted to and can hybridize with, a particular locationon an individual human chromosome. The mapping of relevant sequences tochromosomes according to the present invention is an important firststep in correlating those sequences with gene associated disease. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. Such data are found in, for example, V. McKusick,Mendelian Inheritance in Man (available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (co-inheritance of physicallyadjacent genes). Precise human chromosomal localisations for a genomicsequence (gene fragment etc.) can be determined using Radiation Hybrid(RH) Mapping (Walter, M. Spillett, D., Thomas, P., Weissenbach, J., andGoodfellow, P., (1994) A method for constructing radiation hybrid mapsof whole genomes, Nature Genetics 7, 22-28). A number of RH panels areavailable from Research Genetics (Huntsville, Ala., USA) e.g. theGeneBridge4 RH panel (Hum Mol Genet 1996 March;5(3):339-46 A radiationhybrid map of the human genome. Gyapay G, Schmitt K, Fizames C, Jones H,Vega-Czarny N, Spillett D, Muselet D, Prud'Homme J F, Dib C, Auffray C,Morissette J, Weissenbach J, Goodfellow P N). To determine thechromosomal location of a gene using this panel, 93 PCRs are performedusing primers designed from the gene of interest on RH DNAs. Each ofthese DNAs contains random human genomic fragments maintained in ahamster background (human/hamster hybrid cell lines). These PCRs resultin 93 scores indicating the presence or absence of the PCR product ofthe gene of interest. These scores are compared with scores createdusing PCR products from genomic sequences of known location. Thiscomparison is conducted at http://www.genome.wi.mit.edu/.

[0069] The polynucleotide sequences of the present invention are alsovaluable tools for tissue expression studies. Such studies allow thedetermination of expression patterns of polynucleotides of the presentinvention which may give an indication as to the expression patterns ofthe encoded polypeptides in tissues, by detecting the mRNAs that encodethem. The techniques used are well known in the art and include in situhydridization techniques to clones arrayed on a grid, such as cDNAmicroarray hybridization (Schena et al, Science, 270, 467-470, 1995 andShalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplificationtechniques such as PCR. A preferred method uses the TAQMAN (Trade mark)technology available from Perkin Elmer. Results from these studies canprovide an indication of the normal function of the polypeptide in theorganism. In addition, comparative studies of the normal expressionpattern of mRNAs with that of mRNAs encoded by an alternative form ofthe same gene (for example, one having an alteration in polypeptidecoding potential or a regulatory mutation) can provide valuable insightsinto the role of the polypeptides of the present invention, or that ofinappropriate expression thereof in disease. Such inappropriateexpression may be of a temporal, spatial or simply quantitative nature.

[0070] A further aspect of the present invention relates to antibodies.The polypeptides of the invention or their fragments, or cellsexpressing them, can be used as immunogens to produce antibodies thatare immunospecific for polypeptides of the present invention. The term“immunospecific” means that the antibodies have substantially greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art.

[0071] Antibodies generated against polypeptides of the presentinvention may be obtained by administering the polypeptides orepitope-bearing fragments, or cells to an animal, preferably a non-humananimal, using routine protocols. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique (Kohler, G. and Milstein, C., Nature (1975)256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96,Alan R. Liss, Inc., 1985).

[0072] Techniques for the production of single chain antibodies, such asthose described in U.S. Pat. No. 4,946,778, can also be adapted toproduce single chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

[0073] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or to purify the polypeptidesby affinity chromatography. Antibodies against polypeptides of thepresent invention may also be employed to treat diseases of theinvention, amongst others.

[0074] Polypeptides and polynucleotides of the present invention mayalso be used as vaccines. Accordingly, in a further aspect, the presentinvention relates to a method for inducing an immunological response ina mammal that comprises inoculating the mammal with a polypeptide of thepresent invention, adequate to produce antibody and/or T cell immuneresponse, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said animal from disease, whether thatdisease is already established within the individual or not. Animmunological response in a mammal may also be induced by a methodcomprises delivering a polypeptide of the present invention via a vectordirecting expression of the polynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce antibody to protect said animal from diseases of the invention.One way of administering the vector is by accelerating it into thedesired cells as a coating on particles or otherwise. Such nucleic acidvector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNAhybrid. For use a vaccine, a polypeptide or a nucleic acid vector willbe normally provided as a vaccine formulation (composition). Theformulation may further comprise a suitable carrier. Since a polypeptidemay be broken down in the stomach, it is preferably administeredparenterally (for instance, subcutaneous, intramuscular, intravenous, orintra-dermal injection). Formulations suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions that may contain anti-oxidants, buffers, bacteriostats andsolutes that render the formulation instonic with the blood of therecipient; and aqueous and non-aqueous sterile suspensions that mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

[0075] Polypeptides of the present invention have one or more biologicalfunctions that are of relevance in one or more disease states, inparticular the diseases of the invention hereinbefore mentioned. It istherefore useful to identify compounds that stimulate or inhibit thefunction or level of the polypeptide. Accordingly, in a further aspect,the present invention provides for a method of screening compounds toidentify those that stimulate or inhibit the function or level of thepolypeptide. Such methods identify agonists or antagonists that may beemployed for therapeutic and prophylactic purposes for such diseases ofthe invention as hereinbefore mentioned. Compounds may be identifiedfrom a variety of sources, for example, cells, cell-free preparations,chemical libraries, collections of chemical compounds, and naturalproduct mixtures. Such agonists or antagonists so-identified may benatural or modified substrates, ligands, receptors, enzymes, etc., asthe case may be, of the polypeptide; a structural or functional mimeticthereof (see Coligan et al., Current Protocols in Immunology1(2):Chapter 5 (1991)) or a small molecule. Such small moleculespreferably have a molecular weight below 2,000 daltons, more preferablybetween 300 and 1,000 daltons, and most preferably between 400 and 700daltons. It is preferred that these small molecules are organicmolecules.

[0076] The screening method may simply measure the binding of acandidate compound to the polypeptide, or to cells or membranes bearingthe polypeptide, or a fusion protein thereof, by means of a labeldirectly or indirectly associated with the candidate compound.Alternatively, the screening method may involve measuring or detecting(qualitatively or quantitatively) the competitive binding of a candidatecompound to the polypeptide against a labeled competitor (e.g. agonistor antagonist). Further, these screening methods may test whether thecandidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells bearing the polypeptide. Inhibitors of activation aregenerally assayed in the presence of a known agonist and the effect onactivation by the agonist by the presence of the candidate compound isobserved. Further, the screening methods may simply comprise the stepsof mixing a candidate compound with a solution containing a polypeptideof the present invention, to form a mixture, measuring a ERK-7 activityin the mixture, and comparing the ERK-7 activity of the mixture to acontrol mixture which contains no candidate compound.

[0077] Polypeptides of the present invention may be employed inconventional low capacity screening methods and also in high-throughputscreening (HTS) formats. Such HTS formats include not only thewell-established use of 96- and, more recently, 384-well micotiterplates but also emerging methods such as the nanowell method describedby Schullek et al, Anal Biochem., 246, 20-29, (1997).

[0078] Fusion proteins, such as those made from Fc portion and ERK-7polypeptide, as hereinbefore described, can also be used forhigh-throughput screening assays to identify antagonists for thepolypeptide of the present invention (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

[0079] Screening techniques

[0080] The polynucleotides, polypeptides and antibodies to thepolypeptide of the present invention may also be used to configurescreening methods for detecting the effect of added compounds on theproduction of mRNA and polypeptide in cells. For example, an ELISA assaymay be constructed for measuring secreted or cell associated levels ofpolypeptide using monoclonal and polyclonal antibodies by standardmethods known in the art. This can be used to discover agents that mayinhibit or enhance the production of polypeptide (also called antagonistor agonist, respectively) from suitably manipulated cells or tissues.

[0081] A polypeptide of the present invention may be used to identifymembrane bound or soluble receptors, if any, through standard receptorbinding techniques known in the art. These include, but are not limitedto, ligand binding and crosslinking assays in which the polypeptide islabeled with a radioactive isotope (for instance, 1251), chemicallymodified (for instance, biotinylated), or fused to a peptide sequencesuitable for detection or purification, and incubated with a source ofthe putative receptor (cells, cell membranes, cell supernatants, tissueextracts, bodily fluids). Other methods include biophysical techniquessuch as surface plasmon resonance and spectroscopy. These screeningmethods may also be used to identify agonists and antagonists of thepolypeptide that compete with the binding of the polypeptide to itsreceptors, if any. Standard methods for conducting such assays are wellunderstood in the art.

[0082] Examples of antagonists of polypeptides of the present inventioninclude antibodies or, in some cases, oligonucleotides or proteins thatare closely related to the ligands, substrates, receptors, enzymes,etc., as the case may be, of the polypeptide, e.g., a fragment of theligands, substrates, receptors, enzymes, etc.; or a small molecule thatbind to the polypeptide of the present invention but do not elicit aresponse, so that the activity of the polypeptide is prevented.

[0083] Screening methods may also involve the use of transgenictechnology and ERK-7 gene. The art of constructing transgenic animals iswell established. For example, the ERK-7 gene may be introduced throughmicroinjection into the male pronucleus of fertilized oocytes,retroviral transfer into pre- or post-implantation embryos, or injectionof genetically modified, such as by electroporation, embryonic stemcells into host blastocysts. Particularly useful transgenic animals areso-called “knock-in” animals in which an animal gene is replaced by thehuman equivalent within the genome of that animal. Knock-in transgenicanimals are useful in the drug discovery process, for target validation,where the compound is specific for the human target. Other usefultransgenic animals are so-called “knock-out” animals in which theexpression of the animal ortholog of a polypeptide of the presentinvention and encoded by an endogenous DNA sequence in a cell ispartially or completely annulled. The gene knock-out may be targeted tospecific cells or tissues, may occur only in certain cells or tissues asa consequence of the limitations of the technology, or may occur in all,or substantially all, cells in the animal. Transgenic animal technologyalso offers a whole animal expression-cloning system in which introducedgenes are expressed to give large amounts of polypeptides of the presentinvention

[0084] Screening kits for use in the above described methods form afurther aspect of the present invention. Such screening kits comprise:

[0085] (a) a polypeptide of the present invention;

[0086] (b) a recombinant cell expressing a polypeptide of the presentinvention;

[0087] (c) a cell membrane expressing a polypeptide of the presentinvention; or

[0088] (d) an antibody to a polypeptide of the present invention:

[0089] which polypeptide is preferably that of SEQ ID NO:2.

[0090] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component.

[0091] Glossary

[0092] The following definitions are provided to facilitateunderstanding of certain terms used frequently hereinbefore.“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an

[0093] Fab or other immunoglobulin expression library.

[0094] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

[0095] “Polynucleotide” generally refers to any polyribonucleotide (RNA)or polydeoxribonucleotide (DNA), which may be unmodified or modified RNAor DNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions. single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0096] “Polypeptide” refers to any polypeptide comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, biotinylation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, for instance, ProteinsStructure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H.Freeman and Company, New York, 1993; Wold, F., Post-translationalProtein Modifications: Perspectives and Prospects, 1-12, inPost-translational Covalent Modification of Proteins, B. C. Johnson,Ed., Academic Press. New York, 1983; Seifter et al., “Analysis forprotein modifications and nonprotein cofactors”, Meth Enzymol, 182,626-646, 1990, and Rattan et al., “Protein Synthesis: Post-translationalModifications and Aging”, Ann NY Acad Sci, 663, 48-62, 1992).

[0097] “Fragment” of a polypeptide sequence refers to a polypeptidesequence that is shorter than the reference sequence but that retainsessentially the same biological function or activity as the referencepolypeptide. “Fragment” of a polynucleotide sequence refers to apolynucleotide sequence that is shorter than the reference sequence ofSEQ ID NO:1.

[0098] “Variant” refers to a polynucleotide or polypeptide that differsfrom a reference polynucleotide or polypeptide, but retains theessential properties thereof. A typical variant of a polynucleotidediffers in nucleotide sequence from the reference polynucleotide.Changes in the nucleotide sequence of the variant may or may not alterthe amino acid sequence of a polypeptide encoded by the referencepolynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence from thereference polypeptide. Generally, alterations are limited so that thesequences of the reference polypeptide and the variant are closelysimilar overall and, in many regions, identical. A variant and referencepolypeptide may differ in amino acid sequence by one or moresubstitutions, insertions, deletions in any combination. A substitutedor inserted amino acid residue may or may not be one encoded by thegenetic code. Typical conservative substitutions include Gly, Ala; Val,lle, Leu; Asp, Glu; Asn, Gln; Ser, Thr: Lys, Arg; and Phe and Tyr. Avariant of a polynucleotide or polypeptide may be naturally occurringsuch as an allele, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis. Also included as variants are polypeptides having one or morepost-translational modifications, for instance glycosylation,phosphorylation, methylation, ADP ribosylation and the like. Embodimentsinclude methylation of the N-terminal amino acid, phosphorylations ofserines and threonines and modification of C-terminal glycines.

[0099] “Allele” refers to one of two or more alternative forms of a geneoccurring at a given locus in the genome.

[0100] “Polymorphism” refers to a variation in nucleotide sequence (andencoded polypeptide sequence, if relevant) at a given position in thegenome within a population.

[0101] “Single Nucleotide Polymorphism” (SNP) refers to the occurrenceof nucleotide variability at a single nucleotide position in the genome,within a population. An SNP may occur within a gene or within intergenicregions of the genome. SNPs can be assayed using Allele SpecificAmplification (ASA). For the process at least 3 primers are required. Acommon primer is used in reverse complement to the polymorphism beingassayed. This common primer can be between 50 and 1500 bps from thepolymorphic base. The other two (or more) primers are identical to eachother except that the final 3′ base wobbles to match one of the two (ormore) alleles that make up the polymorphism. Two (or more) PCR reactionsare then conducted on sample DNA, each using the common primer and oneof the Allele Specific Primers.

[0102] “Splice Variant” as used herein refers to cDNA molecules producedfrom RNA molecules initially transcribed from the same genomic DNAsequence but which have undergone alternative RNA splicing. AlternativeRNA splicing occurs when a primary RNA transcript undergoes splicing,generally for the removal of introns, which results in the production ofmore than one mRNA molecule each of that may encode different amino acidsequences. The term splice variant also refers to the proteins encodedby the above cDNA molecules.

[0103] “Identity” reflects a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences,determined by comparing the sequences. In general, identity refers to anexact nucleotide to nucleotide or amino acid to amino acidcorrespondence of the two polynucleotide or two polypeptide sequences,respectively, over the length of the sequences being compared.

[0104] “% Identity”—For sequences where there is not an exactcorrespondence, a “% identity” may be determined. In general, the twosequences to be compared are aligned to give a maximum correlationbetween the sequences. This may include inserting “gaps” in either oneor both sequences, to enhance the degree of alignment. A % identity maybe determined over the whole length of each of the sequences beingcompared (so-called global alignment), that is particularly suitable forsequences of the same or very similar length, or over shorter, definedlengths (so-called local alignment), that is more suitable for sequencesof unequal length.

[0105] “Similarity” is a further, more sophisticated measure of therelationship between two polypeptide sequences. In general, “similarity”means a comparison between the amino acids of two poiypeptide chains, ona residue by residue basis, taking into account not only exactcorrespondences between a between pairs of residues, one from each ofthe sequences being compared (as for identity) but also, where there isnot an exact correspondence, whether, on an evolutionary basis, oneresidue is a likely substitute for the other. This likelihood has anassociated “score” from which the “% similarity” of the two sequencescan then be determined.

[0106] Methods for comparing the identity and similarity of two or moresequences are well known in the art. Thus for instance, programsavailable in the Wisconsin Sequence Analysis Package, version 9.1(Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984, available fromGenetics Computer Group, Madison, Wis., USA), for example the programsBESTFIT and GAP, may be used to determine the % identity between twopolynucleotides and the % identity and the % similarity between twopolypeptide sequences. BESTFIT uses the “local homology” algorithm ofSmith and Waterman (J Mol Biol, 147,195-197, 1981, Advances in AppliedMathematics, 2, 482-489, 1981) and finds the best single region ofsimilarity between two sequences. BESTFIT is more suited to comparingtwo polynucleotide or two polypeptide sequences that are dissimilar inlength, the program assuming that the shorter sequence represents aportion of the longer. In comparison, GAP aligns two sequences, findinga “maximum similarity”, according to the algorithm of Neddleman andWunsch (J Mol Biol, 48, 443-453, 1970). GAP is more suited to comparingsequences that are approximately the same length and an alignment isexpected over the entire length. Preferably, the parameters “Gap Weight”and “Length Weight” used in each program are 50 and 3, forpolynucleotide sequences and 12 and 4 for polypeptide sequences,respectively. Preferably, % identities and similarities are determinedwhen the two sequences being compared are optimally aligned.

[0107] Other programs for determining identity and/or similarity betweensequences are also known in the art, for instance the BLAST family ofprograms (Altschul S F et al, J Mol Biol, 215, 403-410, 1990, Altschul SF et al, Nucleic Acids Res., 25:389-3402, 1997, available from theNational Center for Biotechnology Information (NCBI), Bethesda, Md., USAand accessible through the home page of the NCBI atwww.ncbi.nim.nih.gov) and FASTA (Pearson W R, Methods in Enzymology,183, 63-99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85,2444-2448,1988, available as part of the Wisconsin Sequence AnalysisPackage).

[0108] Preferably, the BLOSUM62 amino acid substitution matrix (HenikoffS and Henikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) isused in polypeptide sequence comparisons including where nucleotidesequences are first translated into amino acid sequences beforecomparison.

[0109] Preferably, the program BESTFIT is used to determine the %identity of a query polynucleotide or a polypeptide sequence withrespect to a reference polynucleotide or a polypeptide sequence, thequery and the reference sequence being optimally aligned and theparameters of the program set at the default value, as hereinbeforedescribed.

[0110] “Identity Index” is a measure of sequence relatedness which maybe used to compare a candidate sequence (polynucleotide or polypeptide)and a reference sequence. Thus, for instance, a candidate polynucleotidesequence having, for example, an Identity Index of 0.95 compared to areference polynucleotide sequence is identical to the reference sequenceexcept that the candidate polynucleotide sequence may include on averageup to five differences per each 100 nucleotides of the referencesequence. Such differences are selected from the group consisting of atleast one nucleotide deletion, substitution, including transition andtransversion, or insertion. These differences may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween these terminal positions, interspersed either individually amongthe nucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. In other words, to obtain apolynucleotide sequence having an Identity Index of 0.95 compared to areference polynucleotide sequence, an average of up to 5 in every 100 ofthe nucleotides of the in the reference sequence may be deleted,substituted or inserted, or any combination thereof, as hereinbeforedescribed. The same applies mutatis mutandis for other values of theIdentity Index, for instance 0.96, 0.97, 0.98 and 0.99.

[0111] Similarly, for a polypeptide, a candidate polypeptide sequencehaving, for example, an Identity Index of 0.95 compared to a referencepolypeptide sequence is identical to the reference sequence except thatthe polypeptide sequence may include an average of up to fivedifferences per each 100 amino acids of the reference sequence. Suchdifferences are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion. These differences may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between these terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. In other words,to obtain a polypeptide sequence having an Identity Index of 0.95compared to a reference polypeptide sequence, an average of up to 5 inevery 100 of the amino acids in the reference sequence may be deleted,substituted or inserted, or any combination thereof, as hereinbeforedescribed. The same applies mutatis mutandis for other values of theIdentity Index, for instance 0.96, 0.97, 0.98 and 0.99.

[0112] The relationship between the number of nucleotide or amino aciddifferences and the Identity Index may be expressed in the followingequation:

n _(a) <x _(a)−(x _(a) ·I),

[0113] in which:

[0114] n_(a) is the number of nucleotide or amino acid differences,

[0115] x_(a) is the total number of nucleotides or amino acids in SEQ IDNO:1 or SEQ ID NO:2, respectively,

[0116] I is the Identity Index,

[0117] · is the symbol for the multiplication operator, and

[0118] in which any non-integer product of x_(a) and I is rounded downto the nearest integer prior to subtracting it from x_(a).

[0119] “Homolog” is a generic term used in the art to indicate apolynucleotide or polypeptide sequence possessing a high degree ofsequence relatedness to a reference sequence. Such relatedness may bequantified by determining the degree of identity and/or similaritybetween the two sequences as hereinbefore defined. Falling within thisgeneric term are the terms “ortholog”, and “paralog”. “Ortholog” refersto a polynucleotide or polypeptide that is the functional equivalent ofthe polynucleotide or polypeptide in another species. “Paralog” refersto a polynucleotideor polypeptide that within the same species which isfunctionally similar.

[0120] “Fusion protein” refers to a protein encoded by two, unrelated,fused genes or fragments thereof. Examples have been disclosed in U.S.Pat. No. 5541087, 5726044. In the case of Fc-ERK-7, employing animmunoglobulin Fc region as a part of a fusion protein is advantageousfor performing the functional expression of Fc- ERK-7 or fragments ofERK-7, to improve pharmacokinetic properties of such a fusion proteinwhen used for therapy and to generate a dimeric ERK-7. The Fc- ERK-7 DNAconstruct comprises in 5′ to 3′ direction, a secretion cassette, i.e. asignal sequence that triggers export from a mammalian cell, DNA encodingan immunoglobulin Fc region fragment, as a fusion partner, and a DNAencoding ERK-7 or fragments thereof. In some uses it would be desirableto be able to alter the intrinsic functional properties (complementbinding, Fc-Receptor binding) by mutating the functional Fc sides whileleaving the rest of the fusion protein untouched or delete the Fc partcompletely after expression.

[0121] All publications and references, including but not limited topatents and patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

1 4 1 1635 DNA Homo sapiens CDS (1)..(1632) 1 atg tgc acc gta gtg gaccct cgc att gtc cgg aga tac cta ctc agg 48 Met Cys Thr Val Val Asp ProArg Ile Val Arg Arg Tyr Leu Leu Arg 1 5 10 15 cgg cag ctc ggg cag ggggcc tat ggc att gtg tgg aag gca gtg gac 96 Arg Gln Leu Gly Gln Gly AlaTyr Gly Ile Val Trp Lys Ala Val Asp 20 25 30 cgg agg act ggt gag gtc gtggcc atc aag aaa atc ttt gat gct ttt 144 Arg Arg Thr Gly Glu Val Val AlaIle Lys Lys Ile Phe Asp Ala Phe 35 40 45 agg gat aag aca gat gcc cag agaaca ttc cgg gaa atc acg ctc ctc 192 Arg Asp Lys Thr Asp Ala Gln Arg ThrPhe Arg Glu Ile Thr Leu Leu 50 55 60 cag gag ttt ggg gac cat ccc aac atcatc agc ctc ctt gac gtg atc 240 Gln Glu Phe Gly Asp His Pro Asn Ile IleSer Leu Leu Asp Val Ile 65 70 75 80 cgg gca gag aac gac agg gac att tacctg gtg ttt gag ttt atg gac 288 Arg Ala Glu Asn Asp Arg Asp Ile Tyr LeuVal Phe Glu Phe Met Asp 85 90 95 act gac ctg aac gca gtc atc cgg aag ggcggc ctg ctg cag gac gtc 336 Thr Asp Leu Asn Ala Val Ile Arg Lys Gly GlyLeu Leu Gln Asp Val 100 105 110 cac gtg cgc tcc atc ttc tac cag ctc ctgcgg gcc acc cgg ttc ctc 384 His Val Arg Ser Ile Phe Tyr Gln Leu Leu ArgAla Thr Arg Phe Leu 115 120 125 cac tcg ggg cac gtt gtg cac cgg gac cagaag ccg tcc aat gtg ctc 432 His Ser Gly His Val Val His Arg Asp Gln LysPro Ser Asn Val Leu 130 135 140 ctg gat gcc aac tgc aca gtg aag ctg tgtgac ttt ggc ctg gcc cgc 480 Leu Asp Ala Asn Cys Thr Val Lys Leu Cys AspPhe Gly Leu Ala Arg 145 150 155 160 tcc ctg ggc gac ctc ccc gag ggg cctgag gac cag gcc gtg aca gag 528 Ser Leu Gly Asp Leu Pro Glu Gly Pro GluAsp Gln Ala Val Thr Glu 165 170 175 tac gtg gcc aca cgc tgg tac cga gcaccg gag gtg ctg ctc tct tcg 576 Tyr Val Ala Thr Arg Trp Tyr Arg Ala ProGlu Val Leu Leu Ser Ser 180 185 190 cac cga tac acc ctt ggg gtg gac atgtgg agt ctg ggc tgt atc ctg 624 His Arg Tyr Thr Leu Gly Val Asp Met TrpSer Leu Gly Cys Ile Leu 195 200 205 ggg gag atg ctg cgg ggg aga ccc ctgttc ccc ggc acg tcc acc ctc 672 Gly Glu Met Leu Arg Gly Arg Pro Leu PhePro Gly Thr Ser Thr Leu 210 215 220 cac cag ctg gag ctg atc ctg gag accatc cca ccg cca tct gag gag 720 His Gln Leu Glu Leu Ile Leu Glu Thr IlePro Pro Pro Ser Glu Glu 225 230 235 240 gac ctc ctg gct ctc ggc tca ggctgc cgt gcc tct gtg ckg cac cag 768 Asp Leu Leu Ala Leu Gly Ser Gly CysArg Ala Ser Val Xaa His Gln 245 250 255 ctg ggg tcc cgg cca cga cag acgctg gat gcc ctc cta ccg cca gac 816 Leu Gly Ser Arg Pro Arg Gln Thr LeuAsp Ala Leu Leu Pro Pro Asp 260 265 270 acc tcc cca gag gcc ttg gac ctcctt agg cga ctc ctg gtg ttc gcc 864 Thr Ser Pro Glu Ala Leu Asp Leu LeuArg Arg Leu Leu Val Phe Ala 275 280 285 ccg gac aag cgg tta agc gcg acccag gca ctg cag cac ccc tac gtg 912 Pro Asp Lys Arg Leu Ser Ala Thr GlnAla Leu Gln His Pro Tyr Val 290 295 300 cag agg ttc cac tgc ccc agc gacgag tgg gca cga gag gca gat gtg 960 Gln Arg Phe His Cys Pro Ser Asp GluTrp Ala Arg Glu Ala Asp Val 305 310 315 320 cgg ccc cgg gca cac gaa ggggtc cag ctc tct gtg cct gag tac cgc 1008 Arg Pro Arg Ala His Glu Gly ValGln Leu Ser Val Pro Glu Tyr Arg 325 330 335 agc cgc gtc tat cag atg atcctg gag tgt gga ggc agc agc ggc acc 1056 Ser Arg Val Tyr Gln Met Ile LeuGlu Cys Gly Gly Ser Ser Gly Thr 340 345 350 tcg aga gag aag ggc ccg gagggt gtc tcc cca agc cag gca cac ctg 1104 Ser Arg Glu Lys Gly Pro Glu GlyVal Ser Pro Ser Gln Ala His Leu 355 360 365 cac aaa ccc aga gcc gac cctcag ctg cct tct agg aca cct gtg cag 1152 His Lys Pro Arg Ala Asp Pro GlnLeu Pro Ser Arg Thr Pro Val Gln 370 375 380 ggt ccc aga ccc agg ccc cagagc agc cca ggc cat gac cct gcc gag 1200 Gly Pro Arg Pro Arg Pro Gln SerSer Pro Gly His Asp Pro Ala Glu 385 390 395 400 cac gag tcc ccc cgt gcagcc aag aac gtt ccc agg cag aac tcc gct 1248 His Glu Ser Pro Arg Ala AlaLys Asn Val Pro Arg Gln Asn Ser Ala 405 410 415 ccc ctg ctc caa act gctctc cta ggg aat ggg gaa agg ccc cct ggg 1296 Pro Leu Leu Gln Thr Ala LeuLeu Gly Asn Gly Glu Arg Pro Pro Gly 420 425 430 gcg aag gaa gcg ccc cccttg aca ctc tcg ctg gtg aag cca agc ggg 1344 Ala Lys Glu Ala Pro Pro LeuThr Leu Ser Leu Val Lys Pro Ser Gly 435 440 445 agg gga gct gcg ccc tccctg acc tcc cag gct gcg gct cag gtg gcc 1392 Arg Gly Ala Ala Pro Ser LeuThr Ser Gln Ala Ala Ala Gln Val Ala 450 455 460 aac cag gcc ctg atc cggggt gac tgg aac cgg ggc ggt ggg gtg agg 1440 Asn Gln Ala Leu Ile Arg GlyAsp Trp Asn Arg Gly Gly Gly Val Arg 465 470 475 480 gtg gcc agc gta caacag gkc cct ccc cgg ctt cct ccg gag gcc cgg 1488 Val Ala Ser Val Gln GlnXaa Pro Pro Arg Leu Pro Pro Glu Ala Arg 485 490 495 ccc ggc cgg agg atgttc agc acc tct gcc ttg cag ggt gcc cag ggg 1536 Pro Gly Arg Arg Met PheSer Thr Ser Ala Leu Gln Gly Ala Gln Gly 500 505 510 ggt gcc agg gct ttgctt gga ggc tac tcc caa gcc tac ggg act gtc 1584 Gly Ala Arg Ala Leu LeuGly Gly Tyr Ser Gln Ala Tyr Gly Thr Val 515 520 525 tgc cac tcg gca ctgggc cac ctg ccc ctg ctg gag ggg cac cat gtg 1632 Cys His Ser Ala Leu GlyHis Leu Pro Leu Leu Glu Gly His His Val 530 535 540 tga 1635 2 544 PRTHomo sapiens MOD_RES (254) variable amino acid 2 Met Cys Thr Val Val AspPro Arg Ile Val Arg Arg Tyr Leu Leu Arg 1 5 10 15 Arg Gln Leu Gly GlnGly Ala Tyr Gly Ile Val Trp Lys Ala Val Asp 20 25 30 Arg Arg Thr Gly GluVal Val Ala Ile Lys Lys Ile Phe Asp Ala Phe 35 40 45 Arg Asp Lys Thr AspAla Gln Arg Thr Phe Arg Glu Ile Thr Leu Leu 50 55 60 Gln Glu Phe Gly AspHis Pro Asn Ile Ile Ser Leu Leu Asp Val Ile 65 70 75 80 Arg Ala Glu AsnAsp Arg Asp Ile Tyr Leu Val Phe Glu Phe Met Asp 85 90 95 Thr Asp Leu AsnAla Val Ile Arg Lys Gly Gly Leu Leu Gln Asp Val 100 105 110 His Val ArgSer Ile Phe Tyr Gln Leu Leu Arg Ala Thr Arg Phe Leu 115 120 125 His SerGly His Val Val His Arg Asp Gln Lys Pro Ser Asn Val Leu 130 135 140 LeuAsp Ala Asn Cys Thr Val Lys Leu Cys Asp Phe Gly Leu Ala Arg 145 150 155160 Ser Leu Gly Asp Leu Pro Glu Gly Pro Glu Asp Gln Ala Val Thr Glu 165170 175 Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu Val Leu Leu Ser Ser180 185 190 His Arg Tyr Thr Leu Gly Val Asp Met Trp Ser Leu Gly Cys IleLeu 195 200 205 Gly Glu Met Leu Arg Gly Arg Pro Leu Phe Pro Gly Thr SerThr Leu 210 215 220 His Gln Leu Glu Leu Ile Leu Glu Thr Ile Pro Pro ProSer Glu Glu 225 230 235 240 Asp Leu Leu Ala Leu Gly Ser Gly Cys Arg AlaSer Val Xaa His Gln 245 250 255 Leu Gly Ser Arg Pro Arg Gln Thr Leu AspAla Leu Leu Pro Pro Asp 260 265 270 Thr Ser Pro Glu Ala Leu Asp Leu LeuArg Arg Leu Leu Val Phe Ala 275 280 285 Pro Asp Lys Arg Leu Ser Ala ThrGln Ala Leu Gln His Pro Tyr Val 290 295 300 Gln Arg Phe His Cys Pro SerAsp Glu Trp Ala Arg Glu Ala Asp Val 305 310 315 320 Arg Pro Arg Ala HisGlu Gly Val Gln Leu Ser Val Pro Glu Tyr Arg 325 330 335 Ser Arg Val TyrGln Met Ile Leu Glu Cys Gly Gly Ser Ser Gly Thr 340 345 350 Ser Arg GluLys Gly Pro Glu Gly Val Ser Pro Ser Gln Ala His Leu 355 360 365 His LysPro Arg Ala Asp Pro Gln Leu Pro Ser Arg Thr Pro Val Gln 370 375 380 GlyPro Arg Pro Arg Pro Gln Ser Ser Pro Gly His Asp Pro Ala Glu 385 390 395400 His Glu Ser Pro Arg Ala Ala Lys Asn Val Pro Arg Gln Asn Ser Ala 405410 415 Pro Leu Leu Gln Thr Ala Leu Leu Gly Asn Gly Glu Arg Pro Pro Gly420 425 430 Ala Lys Glu Ala Pro Pro Leu Thr Leu Ser Leu Val Lys Pro SerGly 435 440 445 Arg Gly Ala Ala Pro Ser Leu Thr Ser Gln Ala Ala Ala GlnVal Ala 450 455 460 Asn Gln Ala Leu Ile Arg Gly Asp Trp Asn Arg Gly GlyGly Val Arg 465 470 475 480 Val Ala Ser Val Gln Gln Xaa Pro Pro Arg LeuPro Pro Glu Ala Arg 485 490 495 Pro Gly Arg Arg Met Phe Ser Thr Ser AlaLeu Gln Gly Ala Gln Gly 500 505 510 Gly Ala Arg Ala Leu Leu Gly Gly TyrSer Gln Ala Tyr Gly Thr Val 515 520 525 Cys His Ser Ala Leu Gly His LeuPro Leu Leu Glu Gly His His Val 530 535 540 3 1063 DNA Homo sapiens CDS(2)..(874) modified_base (1051) a, t, c, g, other or unknown 3 g aga cgacac acg gta gcc cac agg cca cga cag acg ctg gat gcc ctc 49 Arg Arg HisThr Val Ala His Arg Pro Arg Gln Thr Leu Asp Ala Leu 1 5 10 15 cta ccgcca gac acc tcc cca gag gcc ttg gac ctc ctt agg cga cgc 97 Leu Pro ProAsp Thr Ser Pro Glu Ala Leu Asp Leu Leu Arg Arg Arg 20 25 30 ctg gtg ttcgcc ccg gac aag cgg tta agc gcg acc cag gca ctg cag 145 Leu Val Phe AlaPro Asp Lys Arg Leu Ser Ala Thr Gln Ala Leu Gln 35 40 45 cac ccc tac gtgcag agg ttc cac tgc ccc agc gac gag tgg gca cga 193 His Pro Tyr Val GlnArg Phe His Cys Pro Ser Asp Glu Trp Ala Arg 50 55 60 gag gca gat gtg cgcccc ggg cac acg aag ggg tcc agc tct ctg tgc 241 Glu Ala Asp Val Arg ProGly His Thr Lys Gly Ser Ser Ser Leu Cys 65 70 75 80 ctg agt acc gca gccgcg tat att yag atg atc ctg gag tgt gga ggc 289 Leu Ser Thr Ala Ala AlaTyr Ile Xaa Met Ile Leu Glu Cys Gly Gly 85 90 95 agc agc ggc acc tcg agagag aag ggc ccg gag ggt gtc tcc cca agc 337 Ser Ser Gly Thr Ser Arg GluLys Gly Pro Glu Gly Val Ser Pro Ser 100 105 110 cag gca cac ctg cac aaaccc aga gcc gac cct cag ctg cct tct agg 385 Gln Ala His Leu His Lys ProArg Ala Asp Pro Gln Leu Pro Ser Arg 115 120 125 aca cct gtg cag ggt cccaga ccc agg ccc cag agc agc cca ggc cat 433 Thr Pro Val Gln Gly Pro ArgPro Arg Pro Gln Ser Ser Pro Gly His 130 135 140 gac cct gcc gag cac gagtcc ccc cgt gca gcc aag aac gtt ccc agg 481 Asp Pro Ala Glu His Glu SerPro Arg Ala Ala Lys Asn Val Pro Arg 145 150 155 160 cag aac tcc gct cccctg ctc caa act gct ctc cta ggg aat ggg gaa 529 Gln Asn Ser Ala Pro LeuLeu Gln Thr Ala Leu Leu Gly Asn Gly Glu 165 170 175 agg ccc cct ggg gcgaag gaa gcg ccc ccc ttg aca ctc tcg ctg gtg 577 Arg Pro Pro Gly Ala LysGlu Ala Pro Pro Leu Thr Leu Ser Leu Val 180 185 190 aag cca agc ggg agggga gct gcg ccc tcc ctg acc tcc cag gct gcg 625 Lys Pro Ser Gly Arg GlyAla Ala Pro Ser Leu Thr Ser Gln Ala Ala 195 200 205 gct cag gtg gcc aaccag gcc ctg atc cgg ggt gac tgg aac cgg ggc 673 Ala Gln Val Ala Asn GlnAla Leu Ile Arg Gly Asp Trp Asn Arg Gly 210 215 220 ggt ggg gtg agg gtggca gcg tac aac agg gcc ctc ccc ggc ttc ctc 721 Gly Gly Val Arg Val AlaAla Tyr Asn Arg Ala Leu Pro Gly Phe Leu 225 230 235 240 cgg agg ccc ggcccg gcc gga gga tgt tca gca cct ctg cct tgc agg 769 Arg Arg Pro Gly ProAla Gly Gly Cys Ser Ala Pro Leu Pro Cys Arg 245 250 255 gtg ccc agg ggggtg cca ggg ctt tgc ttg gag gct act ccc aag cct 817 Val Pro Arg Gly ValPro Gly Leu Cys Leu Glu Ala Thr Pro Lys Pro 260 265 270 acg gga ctg tctgca ctc ggc act ggg cca cct gcc ctg ctg gag ggg 865 Thr Gly Leu Ser AlaLeu Gly Thr Gly Pro Pro Ala Leu Leu Glu Gly 275 280 285 cac cat gtg tgagccgccgtac tccttacact gccctctgtt ctgcccagcc 917 His His Val 290cttcccagcc cttcagctct gaccccttac cttcctgttg ctgccgtgaa gtcaggactt 977gccgtctctc ggggaccaat aagcctgccc gccaatactc tcataaagca tgtgccaaaa 1037aaaaaaaggg gccnttaaaa ccntta 1063 4 291 PRT Homo sapiens MOD_RES (89)variable amino acid 4 Arg Arg His Thr Val Ala His Arg Pro Arg Gln ThrLeu Asp Ala Leu 1 5 10 15 Leu Pro Pro Asp Thr Ser Pro Glu Ala Leu AspLeu Leu Arg Arg Arg 20 25 30 Leu Val Phe Ala Pro Asp Lys Arg Leu Ser AlaThr Gln Ala Leu Gln 35 40 45 His Pro Tyr Val Gln Arg Phe His Cys Pro SerAsp Glu Trp Ala Arg 50 55 60 Glu Ala Asp Val Arg Pro Gly His Thr Lys GlySer Ser Ser Leu Cys 65 70 75 80 Leu Ser Thr Ala Ala Ala Tyr Ile Xaa MetIle Leu Glu Cys Gly Gly 85 90 95 Ser Ser Gly Thr Ser Arg Glu Lys Gly ProGlu Gly Val Ser Pro Ser 100 105 110 Gln Ala His Leu His Lys Pro Arg AlaAsp Pro Gln Leu Pro Ser Arg 115 120 125 Thr Pro Val Gln Gly Pro Arg ProArg Pro Gln Ser Ser Pro Gly His 130 135 140 Asp Pro Ala Glu His Glu SerPro Arg Ala Ala Lys Asn Val Pro Arg 145 150 155 160 Gln Asn Ser Ala ProLeu Leu Gln Thr Ala Leu Leu Gly Asn Gly Glu 165 170 175 Arg Pro Pro GlyAla Lys Glu Ala Pro Pro Leu Thr Leu Ser Leu Val 180 185 190 Lys Pro SerGly Arg Gly Ala Ala Pro Ser Leu Thr Ser Gln Ala Ala 195 200 205 Ala GlnVal Ala Asn Gln Ala Leu Ile Arg Gly Asp Trp Asn Arg Gly 210 215 220 GlyGly Val Arg Val Ala Ala Tyr Asn Arg Ala Leu Pro Gly Phe Leu 225 230 235240 Arg Arg Pro Gly Pro Ala Gly Gly Cys Ser Ala Pro Leu Pro Cys Arg 245250 255 Val Pro Arg Gly Val Pro Gly Leu Cys Leu Glu Ala Thr Pro Lys Pro260 265 270 Thr Gly Leu Ser Ala Leu Gly Thr Gly Pro Pro Ala Leu Leu GluGly 275 280 285 His His Val 290

1. A polypeptide selected from the group consisting of: (a) apolypeptide encoded by a polynucleotide comprising the sequence of SEQID NO:1; (b) a polypeptide comprising a polypeptide sequence having atleast 95% identity to the polypeptide sequence of SEQ ID NO:2; (c) apolypeptide having at least 95% identity to the polypeptide sequence ofSEQ ID NO:2; (d) the polypeptide sequence of SEQ ID NO:2 and (e)fragments and variants of such polypeptides in (a) to (d).
 2. Thepolypeptide of claim 1 comprising the polypeptide sequence of SEQ IDNO:2.
 3. The polypeptide of claim 1 which is the polypeptide sequence ofSEQ ID NO:2.
 4. A polynucleotide selected from the group consisting of:(a) a polynucleotide comprising a polynucleotide sequence having atleast 95% identity to the polynucleotide sequence of SEQ ID NO:1; (b) apolynucleotide having at least 95% identity to the polynucleotide of SEQID NO:1; (c) a polynucleotide comprising a polynucleotide sequenceencoding a polypeptide sequence having at least 95% identity to thepolypeptide sequence of SEQ ID NO:2; (d) a polynucleotide having apolynucleotide sequence encoding a polypeptide sequence having at least95% identity to the polypeptide sequence of SEQ ID NO:2; (e) apolynucleotide with a nucleotide sequence of at least 100 nucleotidesobtained by screening a library under stringent hybridization conditionswith a labeled probe having the sequence of SEQ ID NO: 1 or a fragmentthereof having at least 15 nucleotides; (f) a polynucleotide which isthe RNA equivalent of a polynucleotide of (a) to (e); (g) apolynucleotide sequence complementary to said polynucleotide of any oneof (a) to (f), and (h) polynucleotides that are variants or fragments ofthe polynucleotides of any one of (a) to (g) or that are complementaryto above mentioned polynucleotides, over the entire length thereof.
 5. Apolynucleotide of claim 4 selected from the group consisting of: (a) apolynucleotide comprising the polynucleotide of SEQ ID NO:l; (b) thepolynucleotide of SEQ ID NO:1; (c) a polynucleotide comprising apolynucleotide sequence encoding the polypeptide of SEQ ID NO:2; and (d)a polynucleotide encoding the polypeptide of SEQ ID NO:2.
 6. Anexpression system comprising a polynucleotide capable of producing apolypeptide of any one of claim 1-3 when said expression vector ispresent in a compatible host cell.
 7. A recombinant host cell comprisingthe expression vector of claim 6 or a membrane thereof expressing thepolypeptide of any one of claim 1-3.
 8. A process for producing apolypeptide of any one of claim 1-3 comprising the step of culturing ahost cell as defined in claim 7 under conditions sufficient for theproduction of said polypeptide and recovering the polypeptide from theculture medium.
 9. A fusion protein consisting of the ImmunoglobulinFc-region and a polypeptide any one of claims 1-3.
 10. An antibodyimmunospecific for the polypeptide of any one of claims 1 to
 3. 11. Amethod for screening to identify compounds that stimulate or inhibit thefunction or level of the polypeptide of any one of claim 1-3 comprisinga method selected from the group consisting of: (a) measuring or,detecting, quantitatively or qualitatively, the binding of a candidatecompound to the polypeptide (or to the cells or membranes expressing thepolypeptide) or a fusion protein thereof by means of a label directly orindirectly associated with the candidate compound; (b) measuring thecompetition of binding of a candidate compound to the polypeptide (or tothe cells or membranes expressing the polypeptide) or a fusion proteinthereof in the presence of a labeled competitior; (c) testing whetherthe candidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells or cell membranes expressing the polypeptide; (d) mixing acandidate compound with a solution containing a polypeptide of any oneof claims 1-3, to form a mixture, measuring activity of the polypeptidein the mixture, and comparing the activity of the mixture to a controlmixture which contains no candidate compound; or (e) detecting theeffect of a candidate compound on the production of mRNA encoding saidpolypeptide or said polypeptide in cells, using for instance, an ELISAassay, and (f) producing said compound according to biotechnological orchemical standard techniques.